Alzheimer's Disease (AD) is a progressive neurodegenerative disease characterised by dementia and associated with the elderly. Currently more than 24.3 million people worldwide have dementia, of whom at least half probably have AD. The incidence of AD is likely to increase as the population ages and this will pose a substantial cost to both careers and healthcare systems. Consequently, efforts to prevent or deter the disease will have substantial benefits.
AD has two main characteristic neuropathological features that represent potential therapeutic targets: a massive over-accumulation in the brain of intracellular protein deposits termed neurofibrillary tangles (NFT) and extracellular protein deposits termed amyloid or senile plaques (SP). The main constituent of SP is β-amyloid (Aβ), a 39-43 amino acid peptide that is cleaved from amyloid precursor protein (APP) and is then released from the cell before accumulating to form SP. NFT, consisting of hyperphosphorylated tau protein, are thought to appear after alterations in APP metabolism. An increase in Aβ, levels, especially the 42 amino acid form (Aβ42), leading eventually to excessive deposition and neurotoxicity in the cortex and limbic system in particular, is thought to be crucial for the pathogenesis of AD.
This amyloid hypothesis is one of the dominant theories of the aetiology of AD.
APP, a type-I integral membrane protein, can be processed into fragments by one of two major proteolytic cleavage pathways. The non-amyloidogenic route involves α-secretase which cleaves within the Aβ region to release soluble sAPPα, a 100 kDa secreted isoform that is thought to possess a trophic function in the CNS, and a C-terminal fragment C83 which is cleaved by γ-secretase to produce P3.
APP exists as three major native forms (alternative splice variants) containing 695, 751 and 770 amino acids. Other mutations produce different forms of APP but these are rare. Conserved in each of these isoforms is the β-secretase cleavage site.
The amyloidogenic pathway involves sequential cleavage of APP by β- and γ-secretases. This route first liberates two peptides after cleavage by β-secretase, sAPPβ and C99 (N-terminal and C-terminal fragments, respectively). The latter, C99, is then further processed by γ-secretase to produce Aβ and the APP C-terminal intracellular domain. β-Secretase is an aspartic protease called β-site APP-cleaving enzyme (BACE, sometimes called BACE1). A homologue of BACE1, BACE2, has also been found, predominantly in the pancreas.
Currently, the only licensed treatments available for AD are symptomatic and target glutaminergic and cholinergic systems. Recently, however, the National Institute for Health and Clinical Excellence (NICE) in the U.K. issued guidelines which state that Reminyl™ (galantamine), Aricept™ (donepezil hydrochloride) and Exelon™ (rivastigmine), all acetylcholinesterase inhibitors originally licensed to treat mild to moderate cases of AD, can now only be prescribed on the National Health Service (NHS) for moderate cases of the disease (NICE, 2006). The glutamate receptor antagonist, Ebixa™ (memantine), previously licensed to treat moderately severe to severe cases of AD in the U.K. has been withdrawn from availability via the NHS (NICE, 2006). These drugs can continue to be prescribed for patients already taking them for mild or moderately severe to severe AD before the guidelines were issued (NICE, 2006). As a result of this new guidance, there are no current licensed treatments for mild AD available on the NHS (Alzheimer's Society, 2007).
Potential further treatments currently being researched include antioxidants and cholesterol-lowering drugs. More recent therapeutic research has targeted Aβ accumulation and potential inhibitors of β- and γ-secretase are being developed. However, as γ-secretase also cleaves Notch, which is crucial for cell development and survival, an inhibitor of γ-secretase would need to be selective. β-secretase knockout can have some deleterious effects in mice (Dominguez et al., 2005), suggesting that inhibition of this enzyme might cause problems. It also has other substrates including APP-like proteins (APPLP) 1 and 2, sialyltransferase ST6Gal 1 and P-selectin glycoprotein ligand 1. Furthermore, an inhibitor would have to be specific for BACE1 over BACE2. Finally, it has been suggested that several cysteine proteases have β-secretase-like activity so inhibiting BACE1 alone may not be sufficient to prevent Aβ production.
Other strategies under investigation include research into the reduction of Aβ aggregation, and an understanding of Aβclearance via various systems such as Aβ degrading enzymes and the ubiquitin-proteosome system.
Other workers have targeted Aβ accumulation by interfering with β-secretase activity, and so effectively inhibiting BACE1, by producing antibodies that bind selectively to the β-secretase site of human APP.
Paganetti et al (2005) co-expressed a form of human APP (APP695) and an ‘intrabody’ consisting of the variable region of an antibody which recognised part of Aβ at a site very close to the β-secretase cleavage site. They observed a stable association between APP and the intrabody which persisted throughout the maturation of APP and resulted in a reduction of intracellular Aβ. Further, not only did this approach protect APP from β-secretase cleavage, it also favoured the alternative cleavage by α-secretase. This resulted in decreased production of the toxic Aβ peptide and the increased production of P3. However, the approach relied on directly transfecting HEK cells with both the intrabody and human APP leading to very high levels of both proteins in their cells. Therapeutically it is very difficult to transfect cells in the brain. Furthermore, even if it were possible to get the DNA into the brain, targeting it to the correct cells and getting the DNA inside the cells would be very difficult. Finally, the intrabody recognises a sequence within the Aβ sequence and may bind to plaques if it leaves the cell. This could trigger an immune response (Citron, 2004) which would be undesirable.
Arbel et al (2005) have also produced antibodies against the β-secretase cleavage site of APP. These workers exploited the cell surface location of APP in order to generate a monoclonal antibody that bound APP at the cell surface prior to being co-internalised into endosomes and thereafter co-existing together through the intracellular trafficking pathways of the cell. In this way a monoclonal antibody specific for the β-secretase cleavage site of APP can be effectively administered and, once bound to its target site, provides sustained inhibition of BACE cleavage of APP. The β-secretase cleavage site resides between amino acids 663 and 671 of APP and comprises the sequence ISEVKMDA (SEQ ID NO: 1) (Arbel et al. 2005). To improve the immunogenicity of their peptide and reduce tolerance to self-antigens, Arbel et al prepared a multiple antigenic peptide (MAP) expressing eight copies of the APP β-secretase cleavage site containing half the Swedish mutation (ISEVKLDA; SEQ ID NO: 2). Using this MAP a monoclonal antibody, designated BBS 1, was identified. Following internalisation into Chinese hamster ovary cells overexpressing human APP, BBS 1 reduced extracellular and intracellular Aβ levels. In addition, the same group injected APP mutant mice with their antibody and showed an improvement in cognition but no effect on Aβ levels (Rakover et al., 2007). Taken together with our own results, these studies suggest that steric hindrance may be a viable method of reducing amyloid production in vivo.
Our own work (Thomas et al 2006) has also shown that it is possible to produce monoclonal antibodies specific for the β-secretase site of APP, in particular monoclonal antibody 2B12. Thus, via steric hindrance, we can inhibit cleavage of APP and so reduce downstream production of Aβ. As with Arbel et al we believe that our monoclonal antibody 2B12 can enter cells by binding to APP when it is at the cell surface and then being internalised when bound to its antigenic protein. However, with 2B12 we were unable to inhibit more than 45% of Aβ40 production and we therefore concluded that perhaps not all APP is trafficked to the cell surface or the less than desirable inhibition was a function of the affinity of the antibody.
However, our subsequent experiments have revealed that it is possible to achieve better results by using a surprisingly active monoclonal antibody that has the ability to bind to APP more efficiently and thus can more effectively inhibit the β-secretase-mediated cleavage of APP. We have called this particularly effective monoclonal antibody 2B3-E10-A8-B10-F10-H3 (2B3).
Notably, although our invention has been described having regard to Alzheimer's Disease it has use in the treatment of conditions characterised by elevated levels of β-amyloid (Aβ 39-43 amino acid peptide) in a subject.